The goals are to elucidate the structures, genomic arrangement, and expression of human transfer RNA genes. Clones of these genes will be isolated by recombinant DNA techniques using 32P-labeled mammalian tRNAs as hybridization probes to screen human gene libraries. The tRNA genes will be characterized by restriction mapping and Southern hybridization techniques, genomic and chromosome-specific blotting experiments, DNA sequence analyses, and transcription studies. Specific aims for this project are: to elucidate the nature of internal promoter and 5'-flanking regulatory elements for the initiation of transcription of human tRNA genes by RNA polymerase III; to isolate human tRNA genes which have intervening sequences and study the post-transcriptional splicing of precursor tRNAs to the mature-sized tRNAs; to study the biosynthesis of human tRNAs by RNA sequence analyses of the various tRNA intermediates generated using human cell-free extracts; and to construct human mutant tRNA genes by in vitro mutagenesis for transcription studies, for the accumulation of altered precursor tRNAs for processing and modification studies, and for studies designed to elucidate the relationships of tRNA primary structure to specific biological functions.